The present invention relates to the field of milk fermentation. More specifically, this invention relates to novel mutants of Streptococcus thermophilus expressing a mutant lactose permease, the lactose transport activity of which is modified. These strains, and ferments comprising them, can be used to obtain fermented dairy products having good conservation properties.
Yogurts are conventionally obtained by fermentation of milk with a combination of various lactic acid bacteria, chosen from strains of Streptococcus thermophilus and of Lactobacillus bulgaricus. During the fermentation, which is carried out at a temperature of approximately 40 to 45° C., these bacteria mainly use lactose as energy substrate, and produce lactic acid which leads to coagulation of the milk; when the pH reaches a value of approximately 4.8 to 4.5, this fermentation step (also known as “acidification”) is terminated by cooling the product. The latter is then kept cold during the rest of the manufacturing and packaging process, until its consumption.
However, cooling does not completely stop the lactic fermentation; even when the product is kept at 4° C., a gradual increase in its acidity is observed over time.
This phenomenon, known as post-acidification, is responsible for degradation of the organoleptic qualities of the product during its storage.
Post-acidification results essentially from the use, by the bacteria, of the lactose remaining in the product at the end of the controlled acidification step. In order to prevent it, it has been proposed to use strains of lactic acid bacteria which do not ferment lactose, or ferment it very little.
The enzymes essential for lactose fermentation in Streptococcus thermophilus and Lactobacillus bulgaricus are encoded by the lactose operon, which contains the lacS gene, encoding lactose permease, and the lacZ gene, encoding β-galactosidase. These proteins are respectively responsible for lactose transport and hydrolysis. In order to obtain non-post-acidifying strains of lactic acid bacteria, it has therefore been proposed to produce artificial variants, or to select natural mutants, in which the activity of at least one of these enzymes is affected.
Patent EP 1078074 (the company Gervais Danone) relates to L. bulgaricus mutants deficient in β-galactosidase activity, comprising nonsense mutation in at least one of the genes of the lactose operon. This patent describes more specifically a mutant for which analysis of the sequence reveals two point mutations: one introducing a stop codon into the β-galactosidase gene, which induces the inability of this mutant to use lactose; the other mutation induces an amino acid change in the permease gene (Lys->Asn at position 122); EP 1078074 does not report any affect of this mutation on the phenotype of the mutant.
WO 01/88150 describes mutants of a Lactobacillus strain. These mutants are incapable of using lactose, but conserve the ability to express β-galactosidase. WO 01/88150 does not specify the nature or the position of the mutation in question, and simply indicates that it may be located in one of the structural genes of the lactose operon, for example the permease, or in a regulatory region of the lactose operon, or in a gene involved in controlling the expression of the lactose operon.
The mutants described in the documents mentioned above have in common the property of being completely incapable of using lactose. They can only grow on milk if the latter is supplemented with a sugar other than lactose, generally glucose. The acidification and post-acidification properties of these mutants are controlled by the amount of glucose added.
In order to provide an alternative to these mutants, the inventors have investigated whether it is possible to obtain strains of lactic acid bacteria having, firstly, an ability to use lactose during their growth that is comparable to that of the wild-type strains, and, secondly, a restricted ability to use lactose during the stationary phase, so as to reduce or abolish the post-acidification phenomenon. With this aim, they were interested in the possibility of acting on the regulation of lactose transport into lactic acid bacterial cells, and in particular into S. thermophilus cells.
The transport of extracellular lactose into S. thermophilus cells is carried out by means of lactose permease LacS. This lactose transport is carried out by symport with a proton, or by antiport with the intracellular galactose resulting from the degradation of the lactose.
The lactose transport is dependent on two phenomena: firstly, the phosphorylation state of the lactose permease and, secondly, the expression of the lacS gene encoding this lactose permease. These two aspects are addressed in detail below.
Phosphorylation of Lactose Permease
The LacS protein is composed of a translocation domain and a regulatory domain (IIA). These domains contain various histidine residues, the phosphorylation of which is involved in the regulation of lactose transport. In particular, the IIA domain can be phosphorylated on histidine 552. This phosphorylation is carried out by the HPr protein (histidine-containing phosphocarrier protein), itself phosphorylated beforehand.
HPr can be phosphorylated:                on a serine by an ATP-dependent protein kinase; the reverse reaction of hydrolysis of HPr(Ser-P) is catalyzed by a phosphatase activity (HPr(Ser-P) phosphatase);        on a histidine HPr(His˜P), with a phosphoryl group originating from phosphoenol pyruvate, and by means of enzyme I (EI).        
Only the HPr form phosphorylated on histidine allows phosphorylation of lactose permease on histidine 552.
It has been observed, on an in vitro model of proteo-liposomes reconstituting the membrane environment of the LacS protein and its phosphorylation by HPr(His˜P), that this phosphorylation has no effect on lactose transport by symport with a proton (Gunnewijk and Poolman, 2000a), but increases by a factor of approximately 2 the flow of lactose/galactose exchange.
Transcription of the lacS Gene
The transcription of the lactose operon is induced by growth in a lactose-containing medium. The promoter of the lacS and Z genes contains a cre (catabolite responsive element) site which allows regulation by CcpA: CcpA represses the expression of lacS and of lacZ. On the other hand, CcpA has an activating effect on transcription of the gene encoding lactate dehydrogenase (van den Bogaard et al., 2000).
The HPr(Ser-P) form is capable of interacting with CcpA. These proteins together will make it possible to form a complex with the cre site, thereby bringing about repression of transcription of the lacS gene (Jones et al., 1997).
It has been observed (Gunnewijk and Poolman, 2000b) that the HPr(Ser-P) form is dominant at the beginning of the exponential growth phase of S. thermophilus cultures and decreases over the course of said phase, whereas the HPr(His˜P) form appears during the exponential phase and is at a maximum at entry into the stationary phase. The change from HPr(Ser-P) to HPr(His˜P) takes place in parallel with the decrease in lactose and the increase in galactose in the culture medium, and with a very large increase in the expression of lactose permease.
Thus, the phosphorylation state of the HPr protein appears to play a role in the regulation of lactose transport, by compensating for the decrease in lactose in the medium, through, firstly, the level of expression of the LacS protein and, secondly, the regulation of its activity.
On the basis of the observations reported above, Gunnewijk and Poolman have proposed the following model: when lactose is abundant in the medium, the expression of the lacS gene is repressed by the HPr(Ser-P)/CcpA complex. During fermentation, the accumulation of galactose in the medium and the decrease in available lactose bring about a decrease in the ability of the bacterium to cause lactose to penetrate (and therefore a decrease in acidification of the medium). This decrease results in a reduction in glycolytic activity, and a decrease in ATP concentration along with an increase in inorganic phosphate concentration, leading to a reduction in the activity of HPr(Ser-P) kinase to the benefit of the activity of HPr(Ser-P) phosphatase, which would have the effect of reducing the concentration of HPr(Ser-P). This reduction in HPr(Ser-P) concentration makes it possible to lift the catabolic repression of the lacS gene and, consequently, to increase the production of lactose permease. In parallel, the increase in HPr(His˜P) would make it possible to increase the phosphorylation of lactose permease on histidine 552, and therefore the ability to transport lactose by antiport with galactose.
This model, which suggests that the phosphorylation of lactose permease on histidine 552 by HPr(His˜P) increases the flow of lactose in cells when the amount of substrate in the medium decreases, makes it possible to suppose that the acidification at the end of the exponential phase could be slowed down if this phosphorylation was prevented. However, it is based in part on in vitro experiments and does not make it possible to judge in advance the real part played in vivo by the increase in the phosphorylation of lactose permease, relative to the increase in its expression, in lactose importation in vivo. In addition, the observations concerning the effects of the concentration of HPr(Ser-P) and HPr(His˜P) on the increase in the expression and in phosphorylation of LacS were made on bacteria in the exponential phase or at the beginning of the stationary phase; no indication is given with regard to the concentrations of these two forms of HPr at more advanced stages of the stationary phase.
The only information available with regard to the effect of the absence of LacS phosphorylation on histidine 552 is given in a publication by Poolman et al. (Poolman et al., 1992), which describes various plasmids containing the sequence encoding the Streptococcus thermophilus LacS enzyme, mutated on various histidine residues. These plasmids are used to transform a strain of E. coli in which the endogenous lacS gene has been previously deleted. The lactose transport in the strains transformed with the various mutants is evaluated in comparison to that observed in the same strain of E. coli containing a plasmid encoding the wild-type LacS enzyme of Streptococcus thermophilus. No significant difference is observed with regard to the H552R mutant in which the natural histidine is replaced with an arginine. Poolman et al. attribute this result either to the ineffectiveness of the phosphorylation of the wild-type LacS enzyme of Streptococcus thermophilus by E. coli HPr(His˜P), or to the fact that this phosphorylation does not play a role in lactose transport.
The inventors have, however, investigated whether a mutation preventing the phosphorylation of LacS on the histidine residue could have an effect on the acidification and post-acidification properties of the mutant bacterium.
For this study, they chose an industrial strain of Streptococcus thermophilus. This strain, deposited with the CNCM on Dec. 12, 2002, under number I-2967, makes it possible to obtain fermented dairy products having an advantageous texture; however, this strain conduces a considerable post-acidification.
The inventors constructed and characterized a mutant of this strain, expressing, in place of the wild-type lactose permease, a mutated lactose permease that cannot be phosphorylated on histidine 552.
They noticed that this mutant strain had an acidification curve different from that of the parent strain. The acidification begins more slowly in the case of the mutant than in that of the parent strain, and the maximum acidification rate of the mutant is lower. However, an equivalent pH is reached after 6 hours of fermentation for the two strains. It is in terms of the post-acidification that the difference between the two strains is the most marked. Under the same storage conditions (28 days of storage at 10° C.), the ΔpH (difference between the pH at D0 and the pH at D28) is of the order of 0.6 in the case of the parent strain, and of the order of 0.4 in the case of the mutant strain. This difference in post-acidification does not come from a difference in terms of the survival of the bacteria. This is in fact equivalent for the two strains. Furthermore, the fermented products obtained with the mutant have the same texture qualities as those obtained with the parent strain.